Malignancies are dependent upon inducing the ingrowth of new blood vessels from the host to supply their oxygen and nutrient needs, and tumor angiogenesis has emerged over the last decade as a promising new target for antitumor therapy. The primary target of tumor-secreted pro- and antiangiogenic factors is the vascular endothelial cell. Our long-term goal is to define the transcriptional regulation of angiogenesis in ECs and use that information to develop new antiangiogenic therapies. Gax is a growth arrest-specific homeobox transcription factor. In vascular smooth muscle in vitro and in vivo, Gax is rapidly downregulated by growth factors and more slowly upregulated by growth arrest stimuli. Its activities include cell cycle arrest via upregulation of p21 expression, and inhibition of migration. In vascular endothelial cells, it also inhibits tube formation. Moreover, Gax expression induces changes in global gene expression consistent with a quiescent phenotype and downregulation of NF-kappaB signaling. These activities suggested to us the hypothesis that Gax is an important transcriptional inhibitor of the angiogenic phenotype in endothelial cells. Consequently, Gax or its downstream targets may represent a promising target for antiangiogenic therapy. We therefore propose these Specific Aims: 1. Identify mechanisms by which Gax regulates EC phenotype by (a) identifying mechanisms that regulate Gax expression in ECs and (b) determining changes in phenotype and global gene expression in ECs that result from modulation of Gax expression. We will examine the effect of both expressing and inhibiting Gax activity in endothelial cells. 2. Determine the mechanism of interaction between Gax expression and the downregulation of NF-kappaB targets in ECs. We will use adenoviral vectors to express Gax in ECs and identify at what point in the NF-kappaB pathway Gax interferes with the activation of NF-kappaB-dependent genes. 3. Determine the function of Gax during in vivo angiogenesis. Gax regulation will be studied during in vivo angiogenesis, and the effect of Gax expression and its inhibition on in vivo angiogenesis will be determined. The results of these studies will significantly enhance our understanding of the transcriptional regulation of endothelial cell phenotypic changes during angiogenesis, characterize a novel interaction between a homeobox gene and NF-kappaB, and provide a basis for designing antiangiogenic therapies that target Gax.